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• W2906700964 abstract "•PfPK9-binding compounds were discovered•PfPK9-binding compounds inhibit K63-linked ubiquitination in Plasmodium•Takinib and PfPK9-selective HS220 inhibit liver-stage Plasmodium•Takinib and PfPK9-selective HS220 increase liver-stage parasite size There is a scarcity of pharmacological tools to interrogate protein kinase function in Plasmodium parasites, the causative agent of malaria. Among Plasmodium's protein kinases, those characterized as atypical represent attractive drug targets as they lack sequence similarity to human proteins. Here, we describe takinib as a small molecule to bind the atypical P. falciparum protein kinase 9 (PfPK9). PfPK9 phosphorylates the Plasmodium E2 ubiquitin-conjugating enzyme PfUBC13, which mediates K63-linkage-specific polyubiquitination. Takinib is a potent human TAK1 inhibitor, thus we developed the Plasmodium-selective takinib analog HS220. We demonstrate that takinib and HS220 decrease K63-linked ubiquitination in P. falciparum, suggesting PfPK9 inhibition in cells. Takinib and HS220 induce a unique phenotype where parasite size in hepatocytes increases, yet high compound concentrations decrease the number of parasites. Our studies highlight the role of PK9 in regulating parasite development and the potential of targeting Plasmodium kinases for malaria control. There is a scarcity of pharmacological tools to interrogate protein kinase function in Plasmodium parasites, the causative agent of malaria. Among Plasmodium's protein kinases, those characterized as atypical represent attractive drug targets as they lack sequence similarity to human proteins. Here, we describe takinib as a small molecule to bind the atypical P. falciparum protein kinase 9 (PfPK9). PfPK9 phosphorylates the Plasmodium E2 ubiquitin-conjugating enzyme PfUBC13, which mediates K63-linkage-specific polyubiquitination. Takinib is a potent human TAK1 inhibitor, thus we developed the Plasmodium-selective takinib analog HS220. We demonstrate that takinib and HS220 decrease K63-linked ubiquitination in P. falciparum, suggesting PfPK9 inhibition in cells. Takinib and HS220 induce a unique phenotype where parasite size in hepatocytes increases, yet high compound concentrations decrease the number of parasites. Our studies highlight the role of PK9 in regulating parasite development and the potential of targeting Plasmodium kinases for malaria control. Intracellular parasites from the genus Plasmodium are the causal agents of malaria, a life-threatening disease that remains a major public health concern globally. Although many advancements have been made to reduce disease burden, over 445,000 deaths occur annually (WHO, 2017WHO World Malaria Report. World Health Organization, 2017Google Scholar) and this number threatens to grow due to the emergence of drug-resistant parasites to first-line treatments (Ashley et al., 2014Ashley E.A. Dhorda M. Fairhurst R.M. Amaratunga C. Lim P. Suon S. Sreng S. Anderson J.M. Mao S. Sam B. et al.Spread of artemisinin resistance in Plasmodium falciparum malaria.N. Engl. J. Med. 2014; 371: 411-423Crossref PubMed Scopus (1442) Google Scholar, Dondorp et al., 2011Dondorp A.M. Fairhurst R.M. Slutsker L. Macarthur J.R. Breman J.G. Guerin P.J. Wellems T.E. Ringwald P. Newman R.D. Plowe C.V. The threat of artemisinin-resistant malaria.N. Engl. J. Med. 2011; 365: 1073-1075Crossref PubMed Scopus (211) Google Scholar, Fairhurst and Dondorp, 2016Fairhurst R.M. Dondorp A.M. Artemisinin-resistant Plasmodium falciparum malaria.Microbiol. Spectr. 2016; 4https://doi.org/10.1128/microbiolspec.EI10-0013-2016Crossref PubMed Scopus (165) Google Scholar). Despite the global impact of malaria, many aspects of Plasmodium biology remain elusive, which hinders the development of novel drugs. In other disease systems, chemical probes used to interrogate protein function have been critical for advancing our understanding of disease biology and facilitating drug development. Protein kinases are important regulators of biological processes in eukaryotes, and thus have been exploited as chemotherapeutic targets in diverse conditions such as cancer, inflammatory diseases, and neurodegenerative disorders (Cohen, 2009Cohen P. Targeting protein kinases for the development of anti-inflammatory drugs.Curr. Opin. Cell Biol. 2009; 21: 317-324Crossref PubMed Scopus (176) Google Scholar, Zhang et al., 2009Zhang J. Yang P.L. Gray N.S. Targeting cancer with small molecule kinase inhibitors.Nat. Rev. Cancer. 2009; 9: 28-39Crossref PubMed Scopus (2067) Google Scholar). The Plasmodium genome encodes 84–99 protein kinases (Anamika et al., 2005Anamika Srinivasan N. Krupa A. A genomic perspective of protein kinases in Plasmodium falciparum.Proteins. 2005; 58: 180-189Crossref PubMed Scopus (141) Google Scholar, Talevich et al., 2011Talevich E. Mirza A. Kannan N. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa.BMC Evol. Biol. 2011; 11: 321Crossref PubMed Scopus (65) Google Scholar, Ward et al., 2004Ward P. Equinet L. Packer J. Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote.BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (404) Google Scholar), and several studies support their critical role for parasite survival at various stages throughout Plasmodium's complex life cycle (Alam et al., 2015Alam M.M. Solyakov L. Bottrill A.R. Flueck C. Siddiqui F.A. Singh S. Mistry S. Viskaduraki M. Lee K. Hopp C.S. et al.Phosphoproteomics reveals malaria parasite protein kinase G as a signalling hub regulating egress and invasion.Nat. Commun. 2015; 6: 7285Crossref PubMed Scopus (115) Google Scholar, Derbyshire et al., 2014Derbyshire E.R. Zuzarte-Luis V. Magalhaes A.D. Kato N. Sanschagrin P.C. Wang J. Zhou W. Miduturu C.V. Mazitschek R. Sliz P. et al.Chemical interrogation of the malaria kinome.Chembiochem. 2014; 15: 1920-1930Crossref PubMed Scopus (26) Google Scholar, McNamara et al., 2013McNamara C.W. Lee M.C. Lim C.S. Lim S.H. Roland J. Nagle A. Simon O. Yeung B.K. Chatterjee A.K. McCormack S.L. et al.Targeting Plasmodium PI(4)K to eliminate malaria.Nature. 2013; 504: 248-253Crossref PubMed Scopus (315) Google Scholar, Siden-Kiamos et al., 2006Siden-Kiamos I. Ecker A. Nyback S. Louis C. Sinden R.E. Billker O. Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion.Mol. Microbiol. 2006; 60: 1355-1363Crossref PubMed Scopus (135) Google Scholar, Solyakov et al., 2011Solyakov L. Halbert J. Alam M.M. Semblat J.P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (262) Google Scholar, Tewari et al., 2010Tewari R. Straschil U. Bateman A. Bohme U. Cherevach I. Gong P. Pain A. Billker O. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission.Cell Host Microbe. 2010; 8: 377-387Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Furthermore, research in the last decade has highlighted the potential for kinases to be drug targets in Plasmodium (Doerig et al., 2005Doerig C. Billker O. Pratt D. Endicott J. Protein kinases as targets for antimalarial intervention: kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes.Biochim. Biophys. Acta. 2005; 1754: 132-150Crossref PubMed Scopus (79) Google Scholar, Lucet et al., 2012Lucet I.S. Tobin A. Drewry D. Wilks A.F. Doerig C. Plasmodium kinases as targets for new-generation antimalarials.Future Med. Chem. 2012; 4: 2295-2310Crossref PubMed Scopus (76) Google Scholar). However, the paucity of small molecules targeting Plasmodium protein kinases is a hindrance to probing their roles in cell signaling and regulation, especially for essential proteins that yield lethal phenotypes upon genetic disruption. In addition, Plasmodium is an obligate intracellular parasite, thus small molecules used for cellular studies require selectivity for the pathogen over host targets. Among the predicted Plasmodium kinases, a particularly intriguing group of atypical kinases are present that are phylogenetically divergent from mammalian kinases (Doerig et al., 2008Doerig C. Billker O. Haystead T. Sharma P. Tobin A.B. Waters N.C. Protein kinases of malaria parasites: an update.Trends Parasitol. 2008; 24: 570-577Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Talevich et al., 2012Talevich E. Tobin A.B. Kannan N. Doerig C. An evolutionary perspective on the kinome of malaria parasites.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367: 2607-2618Crossref PubMed Scopus (47) Google Scholar, Ward et al., 2004Ward P. Equinet L. Packer J. Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote.BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (404) Google Scholar). This sequence divergence presents a unique opportunity to mitigate the challenges of species selectivity. In addition, we predict that some of these atypical kinases are integral to Plasmodium's life stages (Dorin-Semblat et al., 2013Dorin-Semblat D. Bottrill A.R. Solyakov L. Tobin A. Doerig C. Experimental tools for the study of protein phosphorylation in Plasmodium.Methods Mol. Biol. 2013; 923: 241-257Crossref PubMed Google Scholar, Solyakov et al., 2011Solyakov L. Halbert J. Alam M.M. Semblat J.P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (262) Google Scholar, Zhang et al., 2018Zhang M. Wang C. Otto T.D. Oberstaller J. Liao X. Adapa S.R. Udenze K. Bronner I.F. Casandra D. Mayho M. et al.Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.Science. 2018; 360https://doi.org/10.1126/science.aap7847Crossref Scopus (415) Google Scholar), which includes an obligatory, asymptotic stage in the liver and a cyclical stage in red blood cells that causes disease symptoms. Plasmodium falciparum protein kinase 9 (PfPK9) is an essential protein (Solyakov et al., 2011Solyakov L. Halbert J. Alam M.M. Semblat J.P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (262) Google Scholar, Zhang et al., 2018Zhang M. Wang C. Otto T.D. Oberstaller J. Liao X. Adapa S.R. Udenze K. Bronner I.F. Casandra D. Mayho M. et al.Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.Science. 2018; 360https://doi.org/10.1126/science.aap7847Crossref Scopus (415) Google Scholar) that does not cluster with established eukaryotic kinase groups. As a result, PfPK9 is referred to as an orphan kinase among Plasmodium protein kinase families (Solyakov et al., 2011Solyakov L. Halbert J. Alam M.M. Semblat J.P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (262) Google Scholar, Ward et al., 2004Ward P. Equinet L. Packer J. Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote.BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (404) Google Scholar), and the most similar proteins in P. falciparum have only ∼35% sequence identity. To date, the only known target of PfPK9 is the E2 ubiquitin-conjugating enzyme 13 (PfUBC13) (Philip and Haystead, 2007Philip N. Haystead T.A. Characterization of a UBC13 kinase in Plasmodium falciparum.Proc. Natl. Acad. Sci. U S A. 2007; 104: 7845-7850Crossref PubMed Scopus (22) Google Scholar), the homolog of human UBE2N/UBC13 (HsUBC13). HsUBC13 plays a role in DNA repair and immune response pathways by modifying target proteins with K63-linked polyubiquitin chains (Bothos et al., 2003Bothos J. Summers M.K. Venere M. Scolnick D.M. Halazonetis T.D. The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains.Oncogene. 2003; 22: 7101-7107Crossref PubMed Scopus (79) Google Scholar, Brusky et al., 2000Brusky J. Zhu Y. Xiao W. UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae.Curr. Genet. 2000; 37: 168-174Crossref PubMed Scopus (130) Google Scholar, Hodge et al., 2016Hodge C.D. Spyracopoulos L. Glover J.N. Ubc13: the Lys63 ubiquitin chain building machine.Oncotarget. 2016; 7: 64471-64504Crossref PubMed Scopus (53) Google Scholar, Hofmann and Pickart, 1999Hofmann R.M. Pickart C.M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair.Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, Pickart, 2001Pickart C.M. Ubiquitin enters the new millennium.Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), but details about PfPK9 mediation of PfUBC13 remain to be determined. The intriguing role of PfPK9 in Plasmodium biology, its low sequence homology to eukaryotic protein kinases, and compelling genetic evidence that it is essential, makes it an ideal candidate for functional studies. In this study, we characterize the role of PfPK9 in Plasmodium with our discovery that the small molecule takinib binds to the parasite kinase with sub-micromolar affinity. Interestingly, takinib is a potent and selective inhibitor of human TAK1 (HsTAK1) (Totzke et al., 2017Totzke J. Gurbani D. Raphemot R. Hughes P.F. Bodoor K. Carlson D.A. Loiselle D.R. Bera A.K. Eibschutz L.S. Perkins M.M. et al.Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNF-alpha inhibition for cancer and autoimmune disease.Cell Chem. Biol. 2017; 24: 1029-1039.e7Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), which is regulated by HsUBC13 (Hodge et al., 2016Hodge C.D. Spyracopoulos L. Glover J.N. Ubc13: the Lys63 ubiquitin chain building machine.Oncotarget. 2016; 7: 64471-64504Crossref PubMed Scopus (53) Google Scholar). We demonstrate that takinib decreases K63-linked ubiquitination levels in Plasmodium, supporting the role of PfPK9 as a regulator of PfUBC13 in vivo. To achieve species selectivity, 15 takinib analogs were synthesized and characterized, leading to the identification of HS220––a molecule that binds PfPK9 but does not inhibit HsTAK1. Takinib and HS220 inhibit Plasmodium parasite load in hepatoma cells through a unique mechanism that coincides with a disruption of controlled parasite growth. Our discovery and use of PfPK9-targeting molecules represent an important step in understanding biological pathways regulated by this kinase and supports future development of Plasmodium protein kinase inhibitors for malaria control. We established a high-throughput screen to discover small molecules that bind to PfPK9, as no inhibitors of the protein have been previously described. An ATP-competitive binding screen was optimized using GFP-PfPK9 overexpressed in HEK293 cells, which was bound to ATP-Sepharose resin. Binding to the ATP-binding site was evaluated by measuring GFP fluorescence after protein elution in the presence of compound. With this approach, we first examined ATP binding to PfPK9 and determined an apparent Kd (Kd(app)) value for ATP of 0.38 mM (Figure S1A). A Z′ factor of 0.6 was achieved using DMSO as the negative control and 200 mM ATP as the positive control. After this validation, 3,218 compounds from a kinase-targeted library were screened at a single concentration of 500 μM to determine whether they could compete with ATP binding (Figure 1A). The initial screen was performed at 500 μM because the affinity resin has a high immobilized ATP concentration of ∼10 mM. From this primary screen, 304 compounds that produced a ≥2-fold increase in fluorescence intensity relative to the DMSO control were prioritized. Western blot was used as a secondary assay to detect GFP-PfPK9, which confirmed 14 compounds (0.44% hit rate) able to bind to the kinase (Figure 1B). All 14 screening hits were tested for anti-Plasmodium activity and hepatocyte cytotoxicity. Plasmodium assays were completed utilizing the common liver-stage malaria model system, which measures P. berghei parasite load in HepG2 cells via luciferase activity (Derbyshire et al., 2012Derbyshire E.R. Prudencio M. Mota M.M. Clardy J. Liver-stage malaria parasites vulnerable to diverse chemical scaffolds.Proc. Natl. Acad. Sci. U S A. 2012; 109: 8511-8516Crossref PubMed Scopus (103) Google Scholar). PfPK9 is 83% identical to PbPK9 (90% similarity), with particularly high conservation in the ATP-binding pocket, suggesting compounds would bind to both proteins (Figure S1C). When tested at 30 μM, five compounds (1–5) reduced P. berghei parasite load in liver cells by >50% (Figure 2A). HepG2 and HuH7 hepatoma cell viability was assessed to evaluate possible host toxicity and was examined in the same wells as the anti-parasite assays. Similar to other reports, compounds were eliminated for further study if they inhibited liver cell viability ≥50% relative to the DMSO control. When tested at 30 μM, takinib and 2–5 did not decrease HepG2 viability by >50% (Figure S1B). These results suggest that P. berghei inhibition by these compounds is not a consequence of host cytotoxicity. Interestingly, the five compounds with anti-Plasmodium activity represent two different scaffolds, benzimidazoles (takinib, 2–3) and quinolones (4–5). False-positives are a common risk with high-throughput screening campaigns, but the presence of structural analogs among our prioritized hits increases our confidence in their selection for further study. To focus efforts on compounds with cell permeability and anti-Plasmodium activity, takinib and 2–5 were purchased for PfPK9 binding studies. All purchased hits reproduced in the PfPK9 binding assay and were subsequently used to measure binding affinity (Figure 2C). Takinib binding to PfPK9 was determined by converting an acquired half maximal effective concentration (EC50) value to a Kd(app) value with a previously reported equation (Cheng and Prusoff, 1973Cheng Y. Prusoff W.H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12282) Google Scholar, Haystead, 2006Haystead T.A. The purinome, a complex mix of drug and toxicity targets.Curr. Top. Med. Chem. 2006; 6: 1117-1127Crossref PubMed Scopus (49) Google Scholar). With this approach, the takinib Kd(app) value of 0.46 μM was determined. Compounds 2–5 did not saturate binding curves when tested up to 500 μM, suggesting Kd(app) values >0.5 μM. Due to low-micromolar binding affinity, inhibition of Plasmodium liver-stage parasites, and lack of host cytotoxicity, takinib was prioritized for further functional studies. Previous work identified P. falciparum UBC13 (PfUBC13) as a native substrate of PfPK9 by tracking phosphate transfer in P. falciparum-infected erythrocytes (Philip and Haystead, 2007Philip N. Haystead T.A. Characterization of a UBC13 kinase in Plasmodium falciparum.Proc. Natl. Acad. Sci. U S A. 2007; 104: 7845-7850Crossref PubMed Scopus (22) Google Scholar). PfUBC13 is an E2 ubiquitin-conjugating enzyme and a homolog to Homo sapiens UBE2N/UBC13 (HsUBC13), which is the only E2 known to conjugate K63-linked ubiquitin to substrates. We confirmed that PfUBC13 is a PfPK9 substrate in vitro using a radiolabeled ATP kinase assay with purified proteins. In addition to phosphorylating PfUBC13, we found that PfPK9 is capable of phosphorylating HsUBC13 in vitro (Figure S2). To then explore the binding of takinib to PfPK9 in parasites, we monitored changes in K63-linked protein ubiquitination in P. falciparum-infected erythrocytes. The blood-stage Plasmodium model system was selected for this study since there is currently no method to extract Plasmodium parasites from liver cells. Consequently, Plasmodium proteins cannot be deconvoluted from human proteins using commercially available antibodies that detect K63-linked ubiquitin. Blood-stage P. falciparum parasites (ring stage) were treated with 30 μM takinib for 24 hr and K63-linked ubiquitin levels were assessed via western blot with a K63-linkage-specific ubiquitin antibody (anti-K63Ub). We observed that takinib inhibits blood-stage P. falciparum parasites at 100 μM (Figure S3C), therefore we incubated parasites with nonlethal concentrations (≤30 μM) for the ubiquitination study. Compound 5 was also evaluated to determine if a PfPK9-binding compound with a different scaffold similarly affects these post-translational modification (PTM) levels. After incubation with takinib or 5, K63-linked protein ubiquitination in P. falciparum decreased as indicated by reductions in several band intensities relative to the DMSO control (Figure 3A). To further validate downstream inhibition of PfUBC13 activity in parasites by takinib, a dose-response study was completed (0.001–100 μM). We observed that levels of K63-linked ubiquitin in P. falciparum decreased in a takinib-dependent manner (Figure 3B, bands A, C, and D). Interestingly, some protein targets (Figure 3B, band B) were not affected by takinib treatment. As a control, K48-linked ubiquitin levels were concurrently evaluated (Figure S3A) since K48-linked ubiquitin is involved in degradation pathways and is unrelated to the K63-linked ubiquitin signaling cascade. The total intensity of all bands was quantified after staining for K63-linked ubiquitin, K48-linked ubiquitin, and actin (Figure S3B). We observed that the K63-linked ubiquitin signal significantly decreased at both 30 and 100 μM, while no change in K48-linked ubiquitin or actin signals were detected. To investigate the mode of takinib inhibition of Plasmodium liver-stage parasites, dose-response experiments were completed in two hepatocyte cell lines, HepG2 and HuH7. Takinib inhibited P. berghei parasite load in HepG2 and HuH7 cells with EC50 values of 6.7 ± 1.3 and 7.3 ± 0.99 μM, respectively (Figure 4A). To evaluate potential compound cytotoxicity, hepatoma cell viability was assessed under the same experimental conditions as the anti-Plasmodium assays using a fluorescence-based protease activity assay and a luminescence-based assay that detects ATP. Based on these studies, host cell viability does not correlate with parasite load inhibition (Figure 4A). After infection, Plasmodium first migrates through several hepatocytes in a process termed traversal (0–3 hr) before invasion (Mota et al., 2001Mota M.M. Pradel G. Vanderberg J.P. Hafalla J.C. Frevert U. Nussenzweig R.S. Nussenzweig V. Rodriguez A. Migration of Plasmodium sporozoites through cells before infection.Science. 2001; 291: 141-144Crossref PubMed Scopus (398) Google Scholar). To establish if takinib inhibits early (traversal or invasion) or late (transformation and replication) stages of parasite development, we compared its potency when HepG2 cells were treated at the time of infection or after invasion. From these experiments, we observed no significant change in takinib potency (Figure 4B), suggesting that it does not inhibit parasite invasion of host cells. To further probe the biological function of PK9 in Plasmodium, the gene expression profile of PbPK9 was measured throughout liver-stage development. P. berghei-infected HepG2 and HuH7 cells were collected at various hours post-infection (hpi) and analyzed with qRT-PCR. Freshly dissected sporozoites were used to normalize samples collected at 4, 24, and 48 hpi and each time point was normalized to Pb18S to control for parasite numbers, since a single Plasmodium parasite develops into 10,000–30,000 over the course of liver-stage infection (Prudencio et al., 2006Prudencio M. Rodriguez A. Mota M.M. The silent path to thousands of merozoites: the Plasmodium liver stage.Nat. Rev. Microbiol. 2006; 4: 849-856Crossref PubMed Scopus (323) Google Scholar). Pb18S exhibited a time-dependent increase post-infection, indicating parasite maturation and replication (Figure S4A). We observed that PbPK9 is expressed in sporozoites and throughout infection of HepG2 cells (Figure S5A), with the highest levels observed at 4 hpi (Figure 4C). PbPK9 is also expressed throughout infection of HuH7 cells (Figure S4B). To investigate the inhibition of liver-stage Plasmodium parasites by takinib, immunofluorescence assays were performed. P. berghei-infected HuH7 cells were treated with 10 (∼1× EC50) or 30 μM (∼3× EC50) takinib for various periods of time, including 0–24, 24–48, and 0–48 hpi. Infected cells were subsequently labeled with P. berghei HSP70 (PbHSP70) antibody at 48 hpi to visualize the parasites and DAPI to detect nuclei of P. berghei and HuH7 cells. Takinib treatment at 10 μM significantly increased exo-erythrocytic form (EEF) size when compared with DMSO-treated cells at all time periods tested (Figures 5A and 5B ), but the number of EEFs (infected cells) was not affected (Figure 5C). Treatment of P. berghei-infected cells with 30 μM takinib also led to increased EEF size when added for either 0–24 or 24–48 hpi (Figure 5D). Interestingly, the number of EEFs decreased when 30 μM takinib was added for 0–24 or 0–48 hpi (Figure 5E), suggesting events after invasion are influencing the number of parasites. We have previously shown that takinib is a potent inhibitor of HsTAK1 (Totzke et al., 2017Totzke J. Gurbani D. Raphemot R. Hughes P.F. Bodoor K. Carlson D.A. Loiselle D.R. Bera A.K. Eibschutz L.S. Perkins M.M. et al.Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNF-alpha inhibition for cancer and autoimmune disease.Cell Chem. Biol. 2017; 24: 1029-1039.e7Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and, as a consequence, it may bind to PfPK9 or HsTAK1 to inhibit Plasmodium. To discern between these two possibilities, we sought to develop compounds with selectivity for PfPK9 binding over HsTAK1. We synthesized and evaluated a series of 15 takinib analogs for PfPK9 binding using our ATP-competitive binding assay. When screened at 500 and 250 μM (Table S1), seven analogs were found to likely bind to PfPK9 based on relative fluorescence intensity. PfPK9 binding was further confirmed for six of these compounds with western blot (Table S1). Of the validated PfPK9-binding analogs, HS220 and HS230 (Figure 6A) have been previously shown to have little or no effect on HsTAK1 activity (Totzke et al., 2017Totzke J. Gurbani D. Raphemot R. Hughes P.F. Bodoor K. Carlson D.A. Loiselle D.R. Bera A.K. Eibschutz L.S. Perkins M.M. et al.Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNF-alpha inhibition for cancer and autoimmune disease.Cell Chem. Biol. 2017; 24: 1029-1039.e7Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Due to the poor solubility of HS230, HS220 (Figure 6B) was selected for further analysis. Using our competition binding assay, the HS220 Kd(app) of 4.1 ± 0.80 μM was determined for PfPK9 binding (Figure S5A). In previous activity assays, HsTAK1 inhibition was dramatically decreased (160,000-fold less potent) with the change from a primary benzamide (takinib) to a carboxylic acid (HS220) (Totzke et al., 2017Totzke J. Gurbani D. Raphemot R. Hughes P.F. Bodoor K. Carlson D.A. Loiselle D.R. Bera A.K. Eibschutz L.S. Perkins M.M. et al.Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNF-alpha inhibition for cancer and autoimmune disease.Cell Chem. Biol. 2017; 24: 1029-1039.e7Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In contrast, only a minor change in PfPK9 binding affinity (8.9-fold less potent) is observed between HS220 and takinib. To probe takinib/HS220 selectivity in the context of the Plasmodium kinome, their affinity to two unrelated Plasmodium kinases, P. falciparum protein kinase 5 (PfPK5, a cyclin-dependent protein kinase) and P. falciparum calcium-dependent protein kinase 1 (PfCDPK1), was measured. Takinib and HS220 had no affinity for either PfPK5 or PfCDPK1 when tested up to 30 μM (Kd>30 μM, KINOMEScan). This indicates that they do not generally bind to all Plasmodium kinases. The selectivity of HS220 to PfPK9 over HsTAK1 (Figure 6C) supports its use as a chemical probe in cell-based studies. The anti-Plasmodium activity of HS220 was compared with that of takinib. Similar to takinib, HS220 inhibits P. berghei parasite load in HuH7 cells with an EC50 of 43 ± 3.8 μM (Figure 6D). Importantly, no significant change on HuH7 viability was observed upon HS220 treatment. Because of its reduced HsTAK1 affinity, the inhibitory activity of HS220 in parasites supports an essential role for PK9 in Plasmodium, in agreement with genetic studies demonstrating a lethal phenotype after gene disruption (Solyakov et al., 2011Solyakov L. Halbert J. Alam M.M. Semblat J.P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (262) Google Scholar, Zhang et al., 2018Zhang M. Wang C. Otto T.D. Oberstaller J. Liao X. Adapa S.R. Udenze K. Bronner I.F. Casandra D. Mayho M. et al.Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.Science. 2018; 360https://doi.org/10.1126/science.aap7847Crossref Scopus (415) Goog" @default.
• W2906700964 created "2019-01-11" @default.
• W2906700964 creator A5021704786 @default.
• W2906700964 creator A5048744077 @default.
• W2906700964 creator A5050348822 @default.
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• W2906700964 date "2019-03-01" @default.
• W2906700964 modified "2023-09-29" @default.
• W2906700964 title "Plasmodium PK9 Inhibitors Promote Growth of Liver-Stage Parasites" @default.
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